Nitriles are readily converted to the corresponding carboxylic acids by a variety of chemical processes, but these processes typically require strongly acidic or basic reaction conditions and high reaction temperatures, and usually produce unwanted byproducts and/or large amounts of inorganic salts as unwanted waste. Processes in which enzyme-catalyzed hydrolysis converts nitrile-containing substrates to the corresponding carboxylic acids are often preferred to chemical methods because these processes 1) are often run at ambient temperature, 2) do not require the use of strongly acidic or basic reaction conditions, and 3) do not produce large amounts of unwanted byproducts. Especially advantageous over chemical hydrolysis, the enzyme-catalyzed hydrolysis of a variety of aliphatic or aromatic dinitriles can be highly regioselective, where only one of the two nitrile groups is hydrolyzed to the corresponding carboxylic acid ammonium salt.
Enzyme-catalyzed hydrolysis of nitrile substrates to the corresponding carboxylic acids may be accomplished via a one- or two-step reaction (Table 1).
TABLE 1Substrates*Product(s)EnzymeTwo StepReaction 1RCN + H2ORC(O)NH2nitrile hydrataseReaction 2RC(O)NH2 + H2ORC(O)OH + NH3amidaseOne StepReaction 1RCN + 2H2ORC(O)OH + NH3nitrilase*R represents a varying spectrum of organic substituents particular to a chosen enzyme.
A wide variety of bacterial genera collectively possess a diverse spectrum of nitrile hydratase, amidase, or nitrilase activities, including Rhodococcus, Pseudomonas, Alcaligenes, Arthrobacter, Bacillus, Bacteridium, Brevibacterium, Corynebacterium, Agrobacterium, Micrococcus, and Comamonas. Both aqueous suspensions of these microorganisms and the isolated enzymes have been used to convert nitrites to carboxylic acids. Biotechnological use of these enzymes has been recently reviewed by Cowan et al. (Extremophiles (1998) 2:207–216).
A nitrilase enzyme directly converts a nitrite to the corresponding carboxylic acid in aqueous solution without the intermediate formation of an amide. The use of nitrilases for the hydrolysis of aromatic nitrites to the corresponding carboxylic acid ammonium salts has been known for many years, but it is only recently that the use of nitrilases to convert aliphatic nitrites has been reported. Kobayashi et al. (Tetrahedron (1990) 46:5587–5590; J. Bacteriology (1990) 172:4807–4815) have described an aliphatic nitrilase isolated from Rhodococcus rhodochrous K22 which catalyzed the hydrolysis of aliphatic nitrites to the corresponding carboxylic acid ammonium salts; several aliphatic α,ω-dinitriles were also hydrolyzed. A nitrilase from Comamonas testosteroni has been isolated which can convert a range of aliphatic α,ω-dinitriles to either the corresponding ω-cyanocarboxylic acid ammonium salt or the dicarboxylic acid diammonium salt (CA 2,103,616; and Lévy-Schil et al., Gene (1995) 161:15–20).
The nitrilase activity of unimmobilized Acidovorax facilis 72W cells has been used in a process to prepare five-membered or six-membered ring lactams from aliphatic α,ω-dinitriles (U.S. Pat. No. 5,858,736). In that process, an aliphatic α,ω-dinitrile is first converted to an ammonium salt of an ω-cyanocarboxylic acid in aqueous solution using a catalyst having an aliphatic nitrilase (EC 3.5.5.7) activity. The ammonium salt of the ω-cyanocarboxylic acid is then converted directly to the corresponding lactam by hydrogenation in aqueous solution, without isolation of the intermediate ω-cyanocarboxylic acid or ω-amino-carboxylic acid. When the aliphatic α,ω-dinitrile is also unsymmetrically substituted at the α-carbon atom, the nitrilase produces the ω-cyanocarboxylic acid ammonium salt resulting from hydrolysis of the ω-nitrile group with greater than 98% regioselectivity, thereby producing only one of the two possible lactam products during the subsequent hydrogenation. For example, 2-methyl-glutaronitrile (MGN) was hydrolyzed by unimmobilized Acidovorax facilis 72W cells to produce 4-cyanopentanoic acid (4-CPA) ammonium salt with greater than 98% regioselectivity at 100% conversion.
Nitrilase genes have been cloned and expressed in heterologous systems, especially in Escherichia coli. Petre et al. (U.S. Pat. No. 5,635,391) disclose expression of a nitrilase from Comamonas testosteroni in E. coli and Pseudomonas putida. Also disclosed is a method to improve levels of soluble nitrilase protein in E. coli by coexpression of the GroE chaperonin protein, which results in higher nitrilase specific activity. The E. coli promoters Plac and Ptrp were used to drive expression of the nitrilase coding sequences. In E. coli Plac has also been used successfully in expressing nitrilase coding sequences from Alcaligenes faecalis JM3 (Kobayashi et al., Proc. Nat. Acad. Sci. (1993) 90:247 and JP #4-30663), Rhodococcus rhodochrous J1 (Kobayashi et al., J. Biol. Chem. (1992) 267:20746) and Rhodococcus rhodochrous K22 (Kobayashi et al., Biochem. (1992) 31, 9000). Stalker (U.S. Pat. No. 4,810,648) discloses that the gene for a haloarylnitrile-hydrolyzing nitrilase can be expressed under control of its native promoter in E. coli. In U.S. Pat. No. 5,602,014, Mizumura and Yu disclose a specialized regulatory system for expression of nitrilase genes in Rhodococcus erythropolis. 
Nitrilase enzymes are reported to be highly labile and not obtainable in large quantities (Kobayashi et al., Tetrahedron (1990) 46:5587–5590; J. Bacteriology (1990) 172:4807–4815). In contrast to nitrile hydratase, nitrilases are characterized by low specific activities and reaction rates (Nagasawa et al., Appl Microbiol. Biotechnol. (1993) 40:189–195). The inherent thermal instability of nitrile-hydrolyzing enzymes from mesophiles is reported to limit their industrial applications (Cramp et al., Microbiol. (1997) 143:2313–2320).
The problem remains the lack of an industrially useful, thermostable, and highly productive nitrilase enzyme suitable as a catalyst for nitrile-containing substrates in applications (such as the regioselective hydrolysis of aliphatic dinitriles to cyanocarboxylic acids) where high yields of product are obtained under mild reaction conditions (including ambient temperatures and without extreme acidic or basic conditions) and without generating relatively large amounts of undesirable wastes.